Method of determining adenovirus particle concentration
View Patent ↗The invention discloses a simple, accurate and reproducible method of determining viral particle concentration, especially for any serotype of adenovirus. More specifically, an accurate absorptivity/extinction coefficient in units of viral particles per milliliter per absorbance unit per centimeter (vp/mL-AU-cm) for a sample virus, such as adenovirus, at 260 nanometers (nm) is disclosed, as well as an ultraviolet (UV) absorbance method to determine virus particle concentrations based on this established absorptivity/extinction coefficient and absorbances at 260 nm for purified virus preparations, such as adenovirus preparations.
1. A method of determining the virus particle concentration of an adenovirus preparation from a UV absorbance measurement, which comprises:
a) using a previously established absorptivity/extinction coefficient (ε 260 ) for the respective serotype of the adenovirus preparation, wherein said ε 260 is determined under conditions of disruption of adenoviral particles and complete disruption of adenovirus DNA conformation;
b) measuring the A 260 of a sample from the adenovirus preparation which has been prepared under treatment conditions which result in complete disruption of adenoviral particles and complete disruption of adenovirus DNA conformation; and,
c) determining adenoviral particle concentration of said adenoviral sample from the ε 260 and A 260 measurements of step a) and step b).
2. The method of claim 1 wherein the adenovirus serotype is adenovirus 5.
3. The method of claim 1 wherein said previously established absorptivity/extinction coefficient (ε 260 ) is calculated by:
a′) determining the viral particle concentration of a sample from a adenovirus preparation of said adenovirus serotype;
b′) treating an independent sample from said adenovirus preparation under conditions which result in complete disruption of adenoviral particles and complete disruption of adenovirus DNA conformation;
c′) determining the A 260 of the sample from step b′); and,
d′) calculating the ε 260 from the viral particle concentration of step a′), the A 260 value of step c′) and the A 260 value of a matrix blank.
4. The method of claim 1 wherein the treatment conditions for the sample of step b) comprises addition of sodium dodecyl sulfate (SDS) to said adenovirus preparation at a concentration which completely disrupts both adenovirus particles and adenovirus DNA conformation.
5. The method of claim 1 wherein the treatment conditions for the sample of step b) comprises addition of at least about 1% sodium dodecyl sulfate (SDS) followed by incubation at a temperature which results in a complete disruption of both adenovirus particles and adenovirus DNA conformation.
6. The method of claim 3 wherein the treatment conditions for the sample of step b′) comprises addition of sodium dodecyl sulfate (SDS) to said adenovirus preparation at a concentration which completely disrupts both adenovirus particles and adenovirus DNA conformation.
7. The method of claim 3 wherein the treatment conditions for the sample of step b′) comprises addition of at least about 1% sodium dodecyl sulfate (SDS) followed by incubation at a temperature which results in a complete disruption of both adenovirus particles and adenovirus DNA conformation.
8. The method of claim 6 wherein the adenovirus serotype is adenovirus 5.
9. The method of claim 7 wherein the adenovirus serotype is adenovirus 5.